The present results demonstrate the importance of Hsp90 as a modulator of eNOS in APC and IPC in vivo. Isoflurane-preconditioning and IPC significantly decreased myocardial infarct size compared to control experiments, and this protection was abolished by the specific Hsp90 inhibitors, geldanamycin or radicicol. The findings further indicate that Hsp90 association with eNOS is increased by isoflurane in human coronary artery endothelial cells and that enhanced protein-protein interactions contribute to eNOS activation and increased nitric oxide production during APC.
A growing body of evidence implicates eNOS-derived nitric oxide as a critical component of APC signal transduction. We have previously demonstrated that the non-selective NOS inhibitor L-NAME blocked the trigger and mediator phase of delayed APC, whereas specific inhibitors of inducible NOS or neuronal NOS had no effect.19
Endothelial NOS expression (message and protein) was increased immediately and 24 hours after exposure to isoflurane19
and post-conditioning with isoflurane was mediated through eNOS-sensitive regulation of the pro-survival phosphatidylinositol-3-kinase- serine/threonine protein kinase Akt signaling cascade.11
APC with the volatile anesthetic desflurane was also shown to be mediated by eNOS derived nitric oxide.25
APC increased eNOS activity measured in left ventricular myocardium and inhibition of eNOS abolished infarct size reduction when a NOS inhibitor was administered either before or after desflurane.
The current results confirm and extend these previous findings and indicate that APC is critically dependent on both eNOS and Hsp90. Hsp90 is an important physiological regulator of eNOS, and modulation of APC or IPC by Hsp90 has not been previously elucidated. Hsp90 is a highly conserved, mostly cytosolic, protein expressed in all eukaryotic cells26
, and is likely to modulate APC and IPC through an evolutionary conserved cellular response.27
This highly abundant protein is a molecular chaperone involved in protein folding and maturation. Recent evidence indicates that Hsp90 has an integral role in cell signal transduction pathways, and its critical importance in the cell is illustrated by the lethality of its homozygous disruption in Drosophila.28
Hsp90 is a physiologic binding partner and regulator of eNOS,29,30
and impairment of eNOS/Hsp90 interactions disrupts nitric oxide-dependent signaling. Endothelial NOS and Hsp90 form complexes in endothelial and smooth muscle cells in response to a variety of eNOS-activating stimuli. This action enhances phosphorylation of eNOS by serine/threonine protein kinase Akt,31
increases nitric oxide release,2
and facilitates cyclic guanosine monophosphate production.26
In contrast, specific inhibitors of Hsp90, such as geldanamycin and radicicol, affect the conformational state of Hsp90 by binding to its unique adenosine triphosphate binding site32
and prevent agonist-induced eNOS-Hsp90- serine/threonine protein kinase Akt association and eNOS phosphorylation.31
Hsp90/eNOS association may be of importance in ischemic myocardium. Chronic hypoxia increases resistance to myocardial ischemia and reperfusion injury through a nitric oxide-mediated mechanism. This protection is dependent on enhancement of Hsp90/eNOS association and increased nitric oxide production that is blocked by geldanamycin.7
Hsp90 appears to confer a benefit in chronically hypoxic myocardium by promoting nitric oxide generation and limiting superoxide anion production.7
Targeted overexpression of Hsp90 in myocardium reduces infarct size and enhances the association between Hsp90, eNOS and Akt, resulting in increased phosphorylation of eNOS (at Serine 1177).1
In contrast to these beneficial effects, disrupting the interactions between Hsp90 and eNOS in endothelial cells uncouples enzyme activity. As a result of eNOS uncoupling, the enzyme produces superoxide anion and not nitric oxide, and increased superoxide anion production is inhibitable with L-NAME.6,18,33
The present results demonstrate that Hsp90 is a crucial element in IPC and APC in vivo
. Furthermore, experiments conducted in human coronary artery endothelial cells show that isoflurane increases eNOS activity, and that this action is dependent on enhanced association between Hsp90 and eNOS as demonstrated with co-immunoprecipitation and immunohistochemistry. In contrast, isoflurane failed to increase eNOS activity or nitric oxide production in cardiomyocytes. While isoflurane increased nitric oxide concentration in human coronary artery endothelial cells, the concentration of superoxide anion was unaltered. The function of eNOS to generate nitric oxide or superoxide anion is thought to be regulated by site-specific phosphorylation,34
Isoflurane did not change superoxide anion concentrations under baseline conditions, but it is possible that isoflurane might decrease superoxide generation under conditions where eNOS is uncoupled (e.g. during hyperglycemia), and in a manner that is Hsp90-dependent. This hypothesis remains to be tested, however. Isoflurane has been shown to increase the production of signaling reactive oxygen species from mitochondria of cardiomyocytes.36
In contrast, isoflurane had no effect on superoxide anion production from endothelial cells. Thus, it is unlikely that endothelial cells are a source of signaling reactive oxygen species during APC.
The role of eNOS during classical IPC is controversial.14
Prolonged ischemia and reperfusion is associated with the loss of cardiac endothelial NOS protein, and IPC completely prevents loss of NOS protein and increases NOS activity and cyclic guanosine monophosphate levels.37
Genetic models demonstrate a role for eNOS and nitric oxideduring early IPC. Myocardial ischemia and reperfusion injury is attenuated in mice with myocyte-specific overexpression of eNOS38
and infarct size is reduced in transgenic mice overexpressing either bovine or human eNOS.39
Conversely, myocardial infarct size is markedly increased in eNOS knockout mice.40
In the present investigation, infarct size reduction by APC was blocked by L-NAME, but this NOS inhibitor did not abolish the protection of IPC. IPC may be a more robust stimulus for eliciting cardioprotective signaling than APC, and it is possible that a higher concentration of L-NAME might be necessary to block IPC compared to APC. There may also be greater redundancy of signaling pathways in IPC, only one of which includes nitric oxide as a critical intermediate. In contrast to results with L-NAME, inhibitors of Hsp90 blocked both APC and IPC. These results could suggest that Hsp90 may have binding partners in addition to or alternative of eNOS during cardioprotection, and that these are dependent on the specific mechanical (IPC) or pharmacological (APC) stimulus that induces protection. In addition, the role of APC or IPC to enhance Hsp90 association with NOS isoforms in cardiomyocytes is unknown and is the subject of ongoing investigations in our laboratory. Nonetheless, the results indicate that Hsp90 plays an important role to modulate cardioprotection during diverse preconditioning stimuli. Other heat shock proteins, such as Hsp 70, may also contribute to cardioprotection.41
Co-overexpression of Hsp 70 and Hsp 90 increases eNOS protein in endothelial cells, however unlike Hsp90, there is no evidence to indicate that Hsp 70 or other heat shock proteins regulate eNOS coupling.42
The current results should be interpreted within the constraints of several potential limitations. L-NAME produced brief hemodynamic effects during in vivo
experiments that may have theoretically contributed to alterations in myocardial infarct size. However, L-NAME produced similar hemodynamic effects during IPC and APC, yet infarct size reduction was blocked only in the APC group. Thus, it is unlikely that hemodynamic changes alone substantially contributed to the results. However, myocardial oxygen consumption was not directly measured in the current investigation. Experiments were also completed in normal animals and results might be different in diseased43
or aged myocardium.44
The area at risk for infarction is an important determinant of myocardial infarct size in rabbits, but there were no differences in this variable among groups that could account for the current findings. Experiments were completed with two chemically distinct Hsp90 inhibitors. A limitation of the use of geldanamycin in the current investigation is that this drug undergoes oxidation and reduction cycling, and this cycling can produce superoxide anion. In contrast, radicicol does not demonstrate redox cycling, nor does this drug produce reactive oxygen species. Thus, the results cannot be explained by redox-sensitive effects of pharmacological inhibitors. The role of Hsp90 and eNOS during anesthetic preconditioning was investigated using isoflurane. A recent study confirmed the cardioprotective effects of sevoflurane, another halogenated anesthetic, in human endothelial cells.45
Whether Hsp90 is involved in cardioprotection produced by other volatile anesthetic agents is unknown. The effects of isoflurane to modulate Hsp90 interactions and nitric oxide production were examined in human endothelial cells and in mouse HL-1 cardiomyocytes in vitro
. Results may be different in adult rabbit or human cardiomyocytes. Interactions between endothelial cells and cardiomyocytes during cardioprotection are unknown and are an important focus for future investigations.
In conclusion, the current results indicate that Hsp90 has an important function to modulate APC and IPC in vivo. APC enhances the association between Hsp90 and eNOS resulting in activation of this enzyme and enhanced production of nitric oxide in endothelial cells, but not in cardiomyocytes. The results demonstrate that protein-protein interactions are essential to the process of cardioprotection and further suggest a potential role for endothelial cell - cardiomyocyte coupling during APC and IPC.